Nouvelle cible pour le mélanome métastasé

Mount Sinai researchers have identified novel therapeutic targets for metastatic melanoma, according to a study published in Molecular Cell.

The study focused on a gene called AMIGO2 and its partner, called PTK7. Scientists' understanding of AMIGO2's role in cancer has been limited until now, but the researchers discovered that AMIGO2 and PTK7 is required for melanoma cells to grow and survive.

This research also identified a path forward to develop small molecule inhibitors or antibodies against AMIGO2 and PTK7, which are both found on the melanoma cell's membrane. Targeted therapies against PTK7 have already been successfully tested in phase 1 clinical trials for solid tumors, so the groundwork has already been laid in developing similar drugs for melanoma.

"Melanoma is the most aggressive form of skin cancer, affecting more and more patients," according to the study's senior author, Emily Bernstein, PhD, Associate Professor of Oncological Sciences and Dermatology at The Tisch Cancer Institute at the Icahn School of Medicine at Mount Sinai. "While immunotherapy and targeted therapies have significantly improved the outcome for some metastatic melanoma patients, they have had success in a small subset of patients and can cause significant toxic side effects. Thus, their limitations underscore the need for new therapies, highlighting the importance of this research's discovery of novel targets."

The researchers made their discoveries by studying BET proteins, which regulate gene expression in cancer, and their regulation of AMIGO2. When melanoma is growing, the amount of AMIGO2 increases; silencing its function significantly impairs melanoma's growth. In addition, AMIGO2 regulates PTK7 function and PTK7 is also required for melanoma cell survival, so targeting AMIGO2 and PTK7 would also disrupt melanoma's growth.

This study also discovered the potential to identify additional tumor-promoting genes and other therapeutic targets in melanoma by studying other BET target genes.

Early detection is particularly important in cutaneous melanoma, the most aggressive type of skin cancer: with a thickness of little more than one millimetre, the tumour may begin to spread, sending its cells to colonise other organs. When this occurs, the prognosis is usually poor. Treatments have improved considerably, particularly regarding immunotherapy, but melanoma mortality remains very high. One of the important questions to be answered is how melanomas acquire this inherent potential to metastasize. A technique that makes it possible to follow in vivo and, for the first time, very early stages of melanoma progression in mice, is now allowing researchers at the Spanish National Cancer Research Centre (CNIO) to study the process in detail and has even led to the identification of a potential new drug target. The paper is published in the scientific journal, Nature.

The results of the work, which has involved an international team led by the researcher Marisol Soengas at the CNIO, are doubly relevant; according to Soengas: "We have been able to discover unknown mechanisms in the development of melanoma, and to identify new markers of metastasis that we have validated in samples from patients. These results open up new avenues for pharmacological treatments."

One of the novelties of this paper is the development of MetAlert melanoma models. The researchers have developed mouse models that can reveal, without surgery or additional interventions, how melanoma acts throughout the body, even before the occurrence of metastases. The imaging strategy is based on highly innovative work by the group headed by Sagrario Ortega at the CNIO, which used genetic modifications to obtain mice that emit light (bioluminescence) when there is a pathogenic activation of lymphatic vessels. "These bioluminescent mice are ideal for studying melanoma" indicates Ortega, "because the generation of lymphatic vessels, or lymphangiogenesis, is one of the initial steps in the dissemination of this cancer."

The value of MetAlert lies in the fact that it guides researchers when looking for genes and molecules involved in tumour progression at the earliest stage. It also helps study relapses after surgery, or the response to anticancer drugs. To date, the techniques available for use in animals required probes or markers that had to be injected into the area around the tumour, or that were based on the detection of tumour cells once they were already present in other organs, i.e. once metastasis had begun.

As David Olmeda, lead author of the paper, says "one of the major complications in tracking melanomas has been precisely the lack of sensitivity of the standard techniques."

New Mechanisms of Metastasis

The paper now published in Nature details how, thanks to MetAlert, researchers have detected the mechanisms that melanomas activate very early on to create their own pathways of dissemination, in part through the lymph vessels. It has been suspected for some time that, before spreading, melanomas prepare the ground in the organs they are going to colonise. It was believed that this process involved activating the lymphatic vasculature in the tumour first and then in adjacent lymph nodes, the sentinel nodes, in order to reach more distant organs. However, removing the sentinel nodes does not prevent metastases in other organs, which indicates that something is missing in that model.

The CNIO Melanoma group has discovered what it is. Thanks to the MetAlert models, it has been possible to prove that when these tumours are aggressive, they act at a distance much earlier than previously thought, and do so without the need to resort to the proteins that were previously considered essential to activate lymphangiogenesis in the tumour. "These results indicate a change of paradigm in the study of melanoma metastasis," says Soengas.

As a result of their observations, the group decided to get a complete map of the proteins secreted by aggressive and non-aggressive melanomas. The results were instantly clear: "We found many proteins that are secreted specifically by melanomas that act at a distance, but in this paper, we focused on one in particular, MIDKINE, because it was new and could represent an alternative therapeutic target," explains Olmeda.

Midkine: A Key to Melanoma Metastasis and a Marker of Aggressiveness

Once again using the MetAlert mice, the CNIO Melanoma group has shown that MIDKINE plays an essential role in metastasis, to the point that its activation determines the tumour's ability to spread through the body. In addition, they have described an entire set of signals that mediate this process. To this end, CNIO's Melanoma group created MetAlert avatar mice that integrate human tumour samples on their skin. They also generated other MetAlert variants that reproduce mutations that are characteristic of melanomas in humans. The project also included highly sophisticated in vivo microscopy studies carried out in collaboration with the Mount Sinai Icahn School of Medicine in New York.

Following the studies conducted on mouse models, the researchers demonstrated how important MIDKINE would be in patients with melanoma. In collaboration with dermatology and pathology experts at the Hospital 12 de Octubre in Madrid, and the Hospital Clinic in Barcelona, they analysed the expression of MIDKINE in benign lesions (moles) and in melanomas at different stages of development. This experiment demonstrated that patients with high levels of MIDKINE in their lymph nodes have a worse prognosis; a finding that will lead to the use of MIDKINE as a potential biomarker of aggressiveness.

The paper has further implications, because when MIDKINE is inhibited, metastasis is also blocked as the team discovered in animal models.

"In MIDKINE we have found a possible strategy that merits consideration for drug development," says Soengas. "MIDKINE is not the only target, of course, but as melanoma is one of the cancers with the highest number of mutations described, finding a protein that can serve to block metastasis is an important step."

The researchers predict that the discovery of MIDKINE is only the beginning. "These metastasis-visualisation techniques are opening up new avenues of research regarding new tumour mechanisms and other preclinical studies," say Soengas, Ortega, and Olmeda, "and they are very useful because they can be adapted to various types of cancer, not only to melanoma."

Sep. 16, 2013 — Scientists at Sanford-Burnham Medical Research Institute (Sanford-Burnham) today announced the discovery that a gene encoding an enzyme, phosphoinositide-dependent kinase-1 (PDK1), plays an essential role in the development and progression of melanoma. The finding offers a new approach to treating this life-threatening disease

The team of researchers, led by Ze'ev Ronai, Ph.D., professor and scientific director of Sanford-Burnham Medical Research Institute in La Jolla (San Diego, Calif.), used genetic mouse melanoma models to show the importance of the PDK1 gene in melanoma. Specifically, mice lacking the PDK1 gene in their melanocytes (cells that transform to become melanoma) had smaller melanoma tumors, a significant loss of metastasis, and a prolonged survival time. In some cases, the median survival time was increased by more than 50 percent. Further, by treating mice with the PDK1 gene with an inhibitor of PDK1 (PDK1i), the scientists were able to delay the development of melanoma and inhibit metastasis. The published results are available online in the advanced online publication of Oncogene.

"We have shown that PDK1 is required for melanoma metastasis, and that by inactivating the PDK1 enzyme we can delay the onset of melanoma lesions and almost completely abolish metastasis," Ronai said. Prior to this study, it was known that PDK1 activity played an important role in normal cell processes such as cell metabolism, protein translation, and cell survival. PDK1 activity was also known to be associated with specific tumor types. For example, inactivation of PDK1 activity has been shown to inhibit pancreatic cancer. This study provides the first genetic evidence for the importance of PDK1 in melanoma.

David Fisher, M.D., Ph.D., professor and chairman of the Edward Wigglesworth Department of Dermatology, director of the Melanoma Program, and director of Cutaneous Biology at Massachusetts General Hospital, Harvard Medical School, commented, "The study by Ronai and colleagues is novel and important for melanoma therapeutics because it identifies a new and tractable treatment approach. The investigators achieved impressive results which validate PDK1 as a new treatment target for melanoma."

"This collaboration between Sanford-Burnham and Yale researchers shows unequivocally that melanoma cells require PDK1 for both development and metastasis. The team also demonstrates that a molecular inhibitor is capable of duplicating the effects of the genetic approaches suggesting that the cancer field should invest more efforts into PDK1 targets," said Meenhard Herlynn D.V.M., D.Sc., director of Melanoma Research and leader, Molecular and Cellular Oncogenesis program at the Wistar Institute in Philadelphia, Pa.

Melanoma, Disease Progression, and Treatment

Although less common than other types of cancer, melanoma is the most deadly form of skin cancer. In the United States, over 70,000 new cases are diagnosed per year and 9,000 deaths are attributed to the disease. Metastatic melanoma is a progressive form of melanoma that happens when cancerous cells from the original tumor break off, circulate, and form new tumors in other parts of the body, leading to life-threatening disease.

Recently, advances in the treatment of melanoma that activate the immune system by targeting the molecules CTLA4 and PD1, and targeting kinases such as BRAF, have shown promise. Although these drugs have led to improved patient survival, they do not cure melanoma. Therefore, additional therapies are needed. Recently, it has been shown that a combination of targeted therapies can be more effective.

"It is important now to demonstrate the impact of PDK1 inhibition in combination with other therapies currently used in melanoma, including BRAFi or immunological targets (PD1/CTL4A), on melanoma development and metastasis. A number of PDKi are available and others are in development, offering an important addition to the currently available combination therapies. Ultimately, our goal is to see if inhibition of PDK1 will contribute to better outcomes for patients with melanoma," Ronai said.

Mar. 8, 2013 — A multi-institutional study has revealed that BRAF-positive metastatic malignant melanomas develop resistance to treatment with drugs targeting the BRAF/MEK growth pathway through a major change in metabolism. The findings, which will be published in Cancer Cell and have been released online, suggest a strategy to improve the effectiveness of currently available targeted therapies.

"We were surprised to find that melanoma cells treated with the BRAF inhibitor vemurafenib dramatically change the way they produce energy to stay alive," says David E. Fisher, MD, PhD, chief of Dermatology at Massachusetts General Hospital (MGH) and a co-corresponding author of the Cancer Cell paper. "While current BRAF inhibitor treatment is a major improvement -- shrinking tumors in most patients and extending survival for several months -- patients eventually relapse. So there is an ongoing need to improve both the magnitude and durability of these responses."

In about half the cases of malignant melanoma -- the most deadly form of skin cancer -- tumor growth is driven by mutations in the BRAF gene. Research by investigators at the MGH Cancer Center and elsewhere has shown that treatment with drugs that block BRAF activity temporarily halts tumor growth. Combining a BRAF inhibitor with a drug that targets MEK, another protein in the same growth pathway, strengthens and extends the antitumor response. The current study was designed to investigate how BRAF inhibition changes metabolic activity within melanoma cells and to find other possible treatment targets.

The most common way that cells convert glucose into energy is called oxidative phosphorylation and largely relies on the activity of the cellular structures called mitochondria. Many cancer cells use an alternative mechanism that produces the energy compound ATP without involving mitochondria. A series of experiments by the MGH team revealed that the elevated BRAF activity in BRAF-positive melanoma cells suppresses oxidative phosphorylation by reducing expression of a transcription factor called MITF. Suppressing production of MITF reduced levels of a protein called PGC1α that regulates the generation and function of mitochondria. But melanoma cells treated with a BRAF inhibitor showed elevated MITF activity, along with increased expression of oxidative phosphorylation genes and greater numbers of mitochondria. By switching to oxidative phosphorylation to supply the energy they need, the tumor cells increased their ability to survive in spite of BRAF inhibitor treatment.

"These findings suggest that combination treatment with mitochondrial inhibitors could improve the efficacy of BRAF inhibitors in malignant melanoma," says Fisher, the Wigglesworth Professor of Dermatology at Harvard Medical School. "Several small molecules that target mitochondrial metabolism have been identified by investigators here at the MGH and elsewhere, and laboratory investigations of specific combinations of BRAF inhibitors with mitochondrial antagonists are currently underway."

Dec. 19, 2012 — Scientists at The University of Manchester have identified a protein that appears to hold the key to creating more effective drug treatments for melanoma, one of the deadliest cancers.

Researchers funded by Cancer Research UK have been looking at why new drugs called "MEK inhibitors," which are currently being tested in clinical trials, aren't as effective at killing cancer cells as they should be.

They discovered that MITF -- a protein that helps cells to produce pigment but also helps melanoma cells to grow and survive -- is able to provide cancer cells with a resistance to MEK inhibitors.

Dr Claudia Wellbrock and her team at the Wellcome Trust Centre for Cell-Matrix Research compared human melanoma cells that respond to the drug to cells that don't. They discovered that the cells that didn't respond to the drug contained higher levels of the protein SMURF2.

The researchers reduced the level of SMURF2 in the melanoma cancer cells and then treated the tumour with the MEK inhibitor. They found a 100 fold increase in the sensitivity of the cells to the drug. It appears that removing SMURF2 radically decreases the level of MITF in melanoma cells, making the MEK inhibitor a lot more powerful.

Using mice with tumours the team found that over a three week period there was a substantial decrease in tumour growth when the removal of SMURF2 was used in combination with MEK inhibitors.

Dr Wellbrock says: "Much of cancer research is now focussed on finding new drug combinations. It's recognised that cancers frequently find new ways to combat even the most novel and highly efficient drug treatments, so we are now focussing on targeting the mechanisms that allow the cancer cells to overcome the drug effects. We're very excited about the potential for this new approach that has proved to be so effective in our experiments."

One of the drawbacks of the MEK inhibitor drug is that it targets all cells. MEK (MAP/ERK kinase protein) is present in all cells but cancer cells have overactive MEK. This means the drug must be used in small doses and for a lengthy period to avoid harming healthy cells. By reducing SMURF2 to increase the drug's effectiveness smaller doses could be given over a shorter time period, reducing the level of toxicity in healthy cells.

Dr Wellbrock says: "If we can reduce the toxicity to all cells it will mean cancer treatments are less harmful to patients. It's vital that we improve the treatments for melanoma which is the fifth most common cancer in the UK. By the time many people are diagnosed with melanoma the cancer has already started to spread and advanced tumours can be highly resistant to conventional cancer treatments. The development of resistance to new drugs has also been a major drawback. If we can identify more potent and less toxic drug combinations to tackle melanoma then we could save thousands of lives."

This study was funded in part by Cancer Research UK and the results have been published in the Journal of the National Cancer Institute.

Talking about the research Dr Julie Sharp from the charity said: "Recently there have been some really exciting developments in treating melanoma -- but new approaches that tackle the problem of resistance are still needed. This type of research will be a key focus of the planned new Manchester Cancer Research Centre which will bring together a wide range of research expertise to revolutionise cancer treatment."

The next step for Dr Wellbrock will be to find a drug that can reduce the activity of SMURF2 in cancer cells. The Manchester research team are now screening drug libraries for an existing drug that may already be approved for use for a different illness.

It's hoped that identifying a drug to use in combination with MEK inhibitors will provide a much more powerful and ultimately more successful approach to treating melanoma.

Researchers at Moffitt Cancer Center in Tampa, Fla., and colleagues in California have found that the XL888 inhibitor can prevent resistance to the chemotherapy drug vemurafenib, commonly used for treating patients with melanoma.

Vemurafenib resistance is characterized by a diminished apoptosis (programmed cancer cell death) response. According to the researchers, the balance between apoptosis and cell survival is regulated by a family of proteins. The survival of melanoma cells is controlled, in part, by an anti-apoptotic protein (Mcl-1) that is regulated by a particular kind of inhibitor.

Their current findings, tested in six different models of vemurafenib resistance and in both test tube studies and in melanoma patients, demonstrated an induced apoptosis response and tumor regression when the XL888 inhibitor restored the effectiveness of vemurafenib.

The study appeared in a recent issue of Clinical Cancer Research, a publication of the American Association for Cancer Research.

"The impressive clinical response of melanoma patients to vemurafenib has been limited by drug resistance, a considerable challenge for which no management strategies previously existed," said study co-author Keiran S. M. Smalley, Ph.D., of Moffitt's departments of Molecular Oncology and Cutaneous Oncology. "However, we have demonstrated for the first time that the heat shock protein-90 (HSP90) inhibitor XL888 overcomes resistance through a number of mechanisms."

The diversity of resistance mechanism has been expected to complicate the design of future clinical trials to prevent or treat resistance to inhibitors such as vemurafenib.

"That expectation led us to hypothesize that inhibitor resistance might best be managed through broadly targeted strategies that inhibit multiple pathways simultaneously," explained Smalley.

The HSP90 family was known to maintain cancer cells by regulating cancer cells, making it a good target for treatment. According to the authors, the combination of vemurafenib and XL888 overcame vemurafenib resistance by targeting HSP90 through multiple signaling pathways.

Unlike most other cancers, malignant melanoma is disproportionately higher in younger people than in other age groups. More than two young adults (aged 15-34) in the UK are diagnosed with the disease every day.

While survival rates have been improving for the last 25 years and are now amongst the highest for any cancer, malignant melanoma still causes around 46,000 deaths worldwide each year -- around 2,560 of those in the UK. The high death rate is due to cancer cells breaking away from the original tumour and spreading or 'metastasising' to other organs like the brain, causing them to fail. It is its ability to metastasise that makes cancer so dangerous.

Using a grant from the Association for International Cancer Research (AICR), Professor Owen Sansom and his team at the Beatson Institute for Cancer Research have proved that a specific gene (P-Rex1) must be present before malignant melanoma can spread.

In research just published in the scientific journal Nature Communications, Professor Sansom and his colleagues demonstrated the key role that P-Rex1 plays in the spread of malignant melanoma.

P-rex1 joue un rôle dans les métastases.

Using mice models which mirror the common human genetics of melanoma, the researchers found that if P-Rex1 was absent from the cells, the melanoma tumours were unable to spread. Further investigation enabled them to decipher the exact mechanism that P-Rex1 uses to drive metastasis and which is blocked when P-Rex1 is removed.

They then clearly confirmed that human melanoma samples, taken from patients' tumours, contained raised levels of P-Rex1.

Said Professor Sansom: "By contrast P-Rex1 is not present in most other normal human cell types, pointing up its suitability as a gene to be 'switched off' with chemotherapeutic drugs, as there are unlikely to be any unwanted side effects on nearby healthy cells.

"As malignant melanoma is resistant to many forms of chemotherapy, these findings are encouraging. Earlier studies using cancer cell lines implicated P-Rex1 in prostate, breast and ovarian cancer but this is the first time it has been shown to be involved in the metastasis of melanoma in mice models as well as being present at high levels in human tumours and cell lines where it drives invasion into surrounding tissue.

Dr Lara Bennett, scientific communications manager for AICR said Professor Sansom's discovery was an excellent example of how basic research, like that funded by AICR, can help form the building blocks for future treatments.

"Although it is early days and more research is needed, if drugs could be designed to block the effects of P-Rex1, melanoma could be prevented from metastasising," she explained. "This would ensure it remained on the surface of the skin where it could easily be removed through surgery, leading to higher survival rates."

Malignant melanoma incidence rates in Britain have quadrupled over the last thirty years with around 11,760 cases diagnosed in the UK each year and almost 200,000 worldwide.

"If malignant melanoma is caught sufficiently early -- while still only a very thin tumour in the top layers of the skin -- survival rates are much higher," said Dr Bennett.

Researchers have discovered a new way that melanoma cells may become resistant to treatment with vemurafenib (Zelboraf), a targeted therapy that has produced dramatic, if transitory, results for some patients with advanced disease. In some cases, the drug has caused tumors to shrink and even disappear, but the treatment invariably stops working.

By exposing melanoma cells in the laboratory to the drug for extended periods, Dr. David Solit of Memorial Sloan-Kettering Cancer Center and his colleagues have learned that some resistant cells produce a shortened version of the mutant BRAF protein that vemurafenib targets. The shortened protein—which is missing its middle section—is active even in the drug's presence, the researchers reported online November 23 in Nature.

"This is a common mechanism whereby the melanoma tumors overcome the effects of the drug," said Dr. Solit, noting that 6 of 19 patients with resistant tumors had a form of the protein that had been shortened in this way. "We hope that this discovery will lead to more effective treatments."

Vemurafenib blocks growth-promoting signals activated by a mutation in the BRAF gene known as V600E. In August, the Food and Drug Administration (FDA) approved the treatment for patients with unresectable or metastatic melanoma whose tumors have this mutation, which is found in about 60 percent of melanomas.

In their study, Dr. Solit and his colleagues found that the shortened forms of the BRAF V600E protein represent splicing variants—that is, they arose through a change in the processing of the RNA that was transcribed from the gene.

"This is an important study because it identifies the first resistance mechanism to BRAF inhibitors that involves a structural change in BRAF itself," said Dr. Ravi Amaravadi of the Perelman School of Medicine at the University of Pennsylvania, who treats patients with melanoma and was not involved in the study.

It will be important to determine how widespread this resistance mechanism is compared to other proposed resistance mechanisms, Dr. Amaravadi added.

So far, the investigators have found this form of the BRAF V600E protein only in vemurafenib-resistant tumors. They believe that the splicing alteration is specific to BRAF and does not affect splicing in general, suggesting that it could have arisen from a mutation or an epigenetic change.

"Conceptually, we have found a novel form of resistance for any drug," said Dr. Solit. "As happens with other targeted therapies, the drug stops working—but the mechanism is different."

This is an important study because it identifies the first resistance mechanism to BRAF inhibitors that involves a structural change in BRAF itself.

—Dr. Ravi AmaravadiWith drugs such as imatinib (Gleevec) or erlotinib (Tarceva), for example, resistance often occurs when tumors acquire new genetic mutations that prevent a drug from binding to its molecular target. As these resistance mechanisms have been discovered, the knowledge has been used to develop second-generation drugs, such as dasatinib (Sprycel), which can bind to mutant forms of the BCR-ABL kinase to which imatinib cannot bind.

With vemurafenib, the shortened BRAF protein forms complexes within cells that promote growth signals in the presence of the drug. To overcome the resistance, researchers could try to develop more potent drugs and find ways to disrupt the complexes, the authors noted.

Another strategy involves combining drugs that target different proteins, an approach that is already being tried. In June, for example, researchers reported positive preliminary results from an early-phase study testing a BRAF inhibitor in combination with a drug that inhibits a second kinase, MEK, which is part of the same signaling pathway as BRAF. Consequently, the use of a MEK inhibitor could prevent or delay development of a drug-resistant form of BRAF.

"Based on the results of our study, we hypothesize that the combination of the BRAF inhibitor and the MEK inhibitor will be more effective and less toxic than either drug alone," said Dr. Solit. "But we need a randomized study to test this idea."

Studies are also needed to fully characterize the molecular basis for the altered RNA processing that leads to resistance to vemurafenib, as well as to identify additional mechanisms that lead to vemurafenib resistance. If drugs were developed to address the altered RNA processing, these agents could be administered with vemurafenib, Dr. Solit noted.

Dr. Amaravadi agreed. The development of new drugs directed against the molecular changes caused by the BRAF splice variant could "significantly prolong the clinical benefit of BRAF inhibitors," he predicted.

"This study identifies RNA processing as a potentially common resistance mechanism," said co-author Dr. Tom Misteli of NCI's Center for Cancer Research. The findings add to research suggesting the importance of alternative splicing as a mechanism of disease, he added.

"The number of splicing diseases is growing, yet our efforts to target RNA processing as a therapeutic strategy are minimal at the moment," Dr. Misteli said. "RNA therapy offers a largely unexplored, powerful therapeutic strategy."

(Nov. 22, 2011) — Researchers from UNC Lineberger Comprehensive Cancer Center are part of a team that has identified a protein, called P-Rex1, that is key to the movement of cells called melanoblasts. When these cells experience uncontrolled growth, melanoma develops.

Melanoma is one of the only forms of cancer that is still on the rise and is one of the most common forms of cancer in young adults. The incidence of melanoma in women under age 30 has increased more than 50 percent since 1980. Metastases are the major cause of death from melanoma.

The team found that mice lacking the P-Rex1 protein are resistant to melanoma metastases. When researchers tested human melanoma cells and tumor tissue for the protein, P-Rex1 was elevated in the majority of cases -- a clue that the protein plays an important role in the cancer's spread. Their findings were published recently in the journal Nature Communications.

"We know that mutations in a gene called BRAF are important for the development of melanoma and several years ago we published a collaborative paper listing 82 proteins that seem to be affected by this genetic pathway. From that list, we focused on P-Rex1 in collaboration with Dr. Nancy Thomas here at UNC and researchers in the United Kingdom," says Channing Der, PhD, a member of the UNC research team. Der is Kenan Professor of pharmacology at UNC-Chapel Hill and member of UNC Lineberger.

A drug approved this summer, vemurafenib, is the first treatment directed at the BRAF mutation. Clinical trials found that the treatment offers a significant survival benefit.

"As a physician and scientist, I know firsthand the frustration of having very limited therapeutic options to offer to patients with metastatic melanoma," says Nancy Thomas, MD, PhD, whose laboratory analyzed the protein's expression in human cells. "Pinpointing that P-Rex1 plays a key role in metastasis gives us a better understanding of how vemurafenib may work and a target for developing new treatments," she adds.

(Nov. 3, 2011) — Because the incidence of malignant melanoma is rising faster than any other cancer in the U.S., researchers at Moffitt Cancer Center in Tampa, Fla., and colleagues at Tampa-based Intezyne Technologies, Inc., Western Carolina University and the University of Arizona are working overtime to develop new technologies to aid in both malignant melanoma diagnosis and therapy. A tool of great promise comes from the world of nanomedicine -- where tiny drug delivery systems are measured in the billionths of meters and are being designed to deliver targeted therapies.

"Melanoma progression is associated with altered expression of cell surface proteins, including adhesion proteins and receptors," said study co-author David L. Morse, Ph.D., whose work at Moffitt includes experimental therapeutics and diagnostic imaging. "Eighty percent of malignant melanomas express high levels of the MC1R receptor, one of a family of five receptors."

Their study, published in a recent issue of the Journal of Medicinal Chemistry, tested the family of receptors, including MC1R, to find out which receptors would respond best when the right ligand was loaded into a nano-sized spherical delivery device resembling a Koosh Ball called a "micelle."

According to study co-author Robert J. Gillies, Ph.D., director of Molecular and Functional Imaging and vice chair of Radiology Research at Moffitt, MC1R has been in the past investigated as a target for selective imaging and for potential therapeutic agents and is known to play a role in skin pigmentation and hair color. The search for the right "ligand" (a substance that forms a complex with a biomolecule) for use in targeting the right receptor, is ongoing.

"The development of ligands that can be attached to micelles and/or nanoparticles to target cancer cells relative to healthy organs is a subject of great research and great potential," said Gilles.

However, failures in this effort can emerge when attachments lose affinity, when poor stability results in collapse before the nano-sized vehicle gets to the vicinity of the tumor, or when the nanoparticle size is too big to escape the body's vascular system. Each issue needs to be addressed, said Gillies.

In this study, Gilles and Morse and colleagues tested one ligand that was found to have "high affinity and selectivity" for MC1R. That ligand was subsequently modified for attachment to a polymer micelle. Noting the three hurdles to be overcome - ligand affinity, nanoparticle stability and right nanoparticle size - the authors concluded that their chosen ligand "remained selective after attachment" and that the increased binding affinity of the ligand to MC1R demonstrated the stability of the system.

"We are also confident that our micelles are of sufficient size to escape the vasculature, and studies in mice are underway to evaluate the selectivity and stability of this targeted micelle system," concluded Morse.

The Moffitt researchers and their colleagues also feel that this development is a step in the right direction toward more effective imaging of malignant melanoma as well as the development of better targeted therapies for individualized treatment of the disease using nano-sized drug delivery systems.

Zebrafish don't get sunburns, but they can get skin cancer -- at least those fish that have been engineered to model the often deadly human cancer. When Leonard Zon, a Howard Hughes Medical Institute investigator at Children's Hospital Boston, developed this melanoma model five years ago, he hoped to use the tiny striped fish to discover new melanoma genes or new therapies for this aggressive cancer that consistently eludes treatment.

Now, in work described in two papers appearing in the March 24, 2011, issue of the journal Nature, he has done both. Zon and colleagues at multiple institutions used the zebrafish model to discover two new melanoma-promoting proteins that could be targets for therapy. The first, SETDB1, is an enzyme that controls the activity of other genes. It was never suspected of playing a role in cancer, but the new studies show that too much of it can accelerate melanoma.

In a second set of experiments, a team led by Zon examined zebrafish embryos and found that shutting down a protein involved in converting DNA to RNA can block the formation of stem cells that give rise to melanoma. Encouragingly, this can be achieved with an existing arthritis drug. The research also demonstrated this drug works additively with another drug now being evaluated in clinical trials for metastatic melanoma, suggesting that this combination may be a viable option for treating human melanoma.

Melanoma, though still relatively rare, is on the rise in the United States, accounting for 68,000 new cases and 8,700 deaths in 2009. The quest to conquer melanoma begins with trying to understand what turns pigmented cells called melanocytes malicious. In 2002, researchers discovered that about 80 percent of human melanomas have a mutation in the BRAF gene that drives proliferation of the melanocytes. Zon put that gene into zebrafish that also lacked the tumor suppressor gene p53. This made the melanocytes that normally fashion the fish's stripes become darker, splotchy and often cancerous. But benign moles can also have the BRAF mutation, so while it may initiate the process, other genes or pathways must conspire to cause melanoma.

In the first Nature paper, Zon and his 21 coauthors looked for those other genes in a region on human chromosome 1, and found 17 genes that were amplified, or duplicated, in melanoma samples. Having extra copies of a gene can cause cells to overproduce the protein it encodes. When the protein is one that promotes cell growth, too much of it can trigger cancer.

To see if any of the duplicated genes they found accelerated melanoma in zebrafish, Craig Ceol and Yariv Houvras, postdoctoral researchers in Zon's lab, created a handy genetic vehicle, the MiniCoopR, that carried the 17 human genes one-by-one into melanoma-prone zebrafish.

Using "brute force" to analyze more than 2,100 tumors in 3,000-plus fish, the researchers found just one gene, SETDB1, that accelerated melanoma. It made the cancer appear earlier, grow faster, and invade deeper into the muscle and spine. By screening 100 human melanoma cell lines, the team found that SETDB1 was also up-regulated in 70 percent of the tumors.

Extensive biochemicial analysis showed that SETDB1 produces an enzyme that regulates gene expression epigenetically, by modifying the DNA-bearing chromatin in the structure of the chromosome. "What's nice is that enzymes can be inhibited and that could lead to new therapies," says Zon. "Also, the more SETDBI was amplified, the worse the cancer, so we could also potentially use it as a diagnostic marker." Notably, SETDB1 is also over-expressed in ovarian, breast, and liver cancers.

For the second paper, Zon and 19 coauthors, including HHMI investigator Sean Morrison at the University of Michigan, examined the embryos of the zebrafish that were genetically modified to be prone to developing melanoma. The embryos appeared normal, but by analyzing their gene expression, Richard White, a postdoctoral researcher in Zon's lab, discovered a set of 127 genes that are mis-expressed in melanomas and that predict which fish will get melanoma.

Notably, these genes are important to a specialized stem/progenitor cell called the embryonic neural crest cell, which differentiates during development and gives rise to melanocytes, facial bones, and connective tissues. White showed that there were more of those cells than normal in the melanoma-prone embryos. "Essentially, these fish had extra stem cells," Zon says, "which perhaps makes the cancer more aggressive."

The team then screened 2,000 chemicals until they found one that perturbed the development of these neural crest cells. "We didn't have any idea what this molecule did," recalls Zon, but a database suggested that the molecule could block an enzyme called DHODH, or dihydroorotate dehydrogenase. The database also suggested that a drug used to treat rheumatoid arthritis drug, leflunomide, would have the same effect.

The scientists found that DHODH, an enzyme used to produce uridine (one of the RNA nucleic bases), is involved in transcribing DNA into RNA -- an essential step before the protein encoded by the gene can be manufactured. "We couldn't imagine how an enzyme important to every cell could be relevant to human melanoma or the development of the neural crest lineage," Zon says. "But we went further with a series of quite surprising experiments in zebrafish and human melanoma cell lines and discovered that leflunomide selectively blocks transcriptional elongation in melanocyte and neural crest genes. We don't know why inhibiting DHODH just stops these genes in their tracks, but that change in expression changes the cell's fate. The cell no longer thinks that it's the neural crest cell or a melanocyte. The drug takes the melanocyte out of the melanoma." But other cells and tissues were less affected by the drug.

After determining that leflunomide slowed the growth of tumors in mice engrafted with human melanoma, Zon's group combined it with a BRAF inhibitor made by Plexxikon. That drug is now in late stage clinical trials for metastatic melanoma. "In these trials, some tumors just melt away. But unfortunately the cancer usually returns after about six months," Zon says. "We thought combining that drug, which targets a specific oncogenic mutation, with leflunomide, which changes the cell's lineage, could have a beneficial effect."

It did, at least in mice. Compared to each drug alone, the combination led to a marked decrease in melanoma, and even with low doses of each drug the tumors went away completely in 40 percent of the mice. This drug combination may enter human clinical trials within a year, and it may be possible to use lower doses of each drug to decrease the risk of side effects and resistance.

Zon says the zebrafish is the ideal model for doing mechanistic studies that link genomics and biochemistry to understand how cancers occur in vivo and how to prevent them. "You need thousands of animals, and you would need an entire mouse facility to do what we can do with a few 50-gallon fish tanks." Also fish embryos grow externally, so researchers can plunk five embryos into each well of 96-well plates to screen thousands of chemicals to discover drug candidates.

Interestingly, some of genes that leflunomide pauses are those controlled by myc, a well known oncogene implicated in several cancer types. Recent work shows that myc controls transcription elongation in embryonic stem cells, and the zebrafish work now highlights myc's importance in melanoma as well. So as with the SETDB1 discovery, this finding may have relevance to other cancers -- and the zebrafish tank will be the perfect place to find out.

New data generated in vitro using human melanoma cell lines and resected tumors by Norman Sharpless and colleagues at the University of North Carolina School of Medicine, Chapel Hill, has identified one mechanism that represses DC function in MM.

Expression of CD200 mRNA and protein was found to be higher in resected human melanomas than in other solid tumors. Further analysis revealed that expression of CD200 was regulated by the N-RAS/B-RAF/MEK/ERK MAP kinase signaling pathway, which is aberrantly activated in approximately 80% of individuals with MM.

In vitro analysis indicated the potential functional significance of high levels of CD200 expression -- it enabled melanoma cell lines to repress activation of antitumor T cell immune responses by DCs. The authors therefore suggested that targeting the interaction between CD200 and its receptor might provide a new strategy for the treatment of MM.